I purified an MBP fusion from yeast using a simple Tris-based elution buffer and denatured in SDS-PAGE buffer. Another protein co-purified with it. I noticed that heating at 95° caused this protein to smear upwards from its normal gel mobility. However heating at 37° did not cause smearing. The smear is not oxidative cross linking (its not reversible with DTT). Is this the maltose in the buffer reacting with the protein? anyone else seen this effect when heating MBP eluates? Is there a way to avoid it (other than using lower temp, which for technical reasons, I can't do).
Related question: can I use less than 10 mM maltose to elute MBP fusions? would 1 mM work?
Hi Colin,
Maltose is a reducing sugar, so heating it in the presence of proteins can lead to Maillard's reaction which translates in proteins-sugars aggregates. To overcome this problem I would suggest that you proceed to a buffer exchange with a maltose free buffer before conditioning your sample for SDS-PAGE.
Good luck
Thanks, i had suspected mailard reaction too. Is this something you have personally encountered, or just hypothetical?
I did not experienced it personally so you can call it an educated guess. If you manage to get rid of maltose before loading your gel you'll have your answer.
- Badges
- Science method